The present invention pertains to improved methods of using dye-ligand affinity chromatography for the isolation or purification of antibodies or proteins containing an antibody fragment (such as Fc fusion proteins) from a mixture of undesirable contaminants (also known as PRIs or “product related impurities”).
Monoclonal antibodies and Fc fusion proteins are being produced in cell culture in ever increasing quantities. With this comes the greater downstream challenge of maintaining sufficient resin capacity while being able to remove product-related impurities (PRIs; e.g., aggregates, half-antibodies, misfolded antibody forms). Traditional Protein-A purification processes employ a costly capture step to remove host cell proteins and DNA with little removal of PRIs, thus shifting the burden downstream to removal of PRIs (e.g., using HIC, SEC), often with substantial loss of yield. Removal of these PRIs can be the most challenging aspect of the purification process due to their higher starting quantities, their biochemical similarity to the product of interest, and their enrichment on Protein-A chromatography media. Alternative approaches to capture can be done at reduced cost but purity is often sacrificed. The ideal process thus would be a low cost capture step that delivers a product of high purity, yield and low PRI content.
Methods of using a low cost, non-traditional chromatography resin (a dye-ligand chromatography resin) was successfully developed for the capture of Fc-containing proteins at loading capacities comparable to Protein A resins. Optimizations of wash and elution steps with mobile phase modifiers resulted in significant removal of PRIs while maintaining high product purity and yield.
Dye-Ligand Affinity Chromatography
Dye-ligand affinity chromatography is a “mixed-mode” chromatographic media because such media rely on two separate forces to bind target molecules. In the case of dye-ligand affinity media these two forces are cation exchange and hydrophobic interactions. See FIG. 13.
For additional information on conventional methods in dye-ligand affinity chromatography there are numerous publications readily available and understood by those of ordinary skill in the art. See, for example:                Handbook of Affinity Chromatography by David S. Hage, 2nd Edition, 2006, Chromatographic Science Series, vol. 92, Chapter 9, “Dye-Ligand and Biomimetic Affinity Chromatography” (CRC Press, 2006, ISBN 0824740572 and 9780824740573);        Dye-ligand chromatography for the resolution and purification of restriction endonucleases, Journal Applied Biochemistry and Biotechnology, Vol. 15, No. 3 (October, 1987), published by Humana Press Inc. (ISSN 0273-2289 (print) 1599-0291 (online);        Dye-ligand affinity chromatography: the interaction of Cibacron Blue F3GA with proteins and enzymes, by S. Subramanian S., CRC Crit Rev Biochem., 16(2): 169-205 (1984);        Affinity chromatography on immobilised triazine dyes-Studies on the interaction with multinucleotide-dependent enzymes, by Y. D. Clonis and C. R. Lowe CR., Biochim Biophys Acta., 659(1): 86-98 (1981);        Triazine-dye affinity; chromatography, by T. Atkinson, et al., Biochem Soc Trans., 9(4): 290-3 (1981);        The applications of reactive dyes in enzyme and protein downstream processing, Y. D. Clonis, Crit Rev Biotechnol, 7(4): 263-79 (1988);        Chromatography on immobilized reactive dyes, by E. Stellwagen, Methods Enzymol., 182: 343-57 (1990);        Dye-affinity techniques for bioprocessing: recent developments, N. Garg, et al., J Mol Recognit, 9(4): 259-74 (1996);        Dye-ligand affinity systems, by A. Denizli and E. Piskin, J Biochem Biophys Methods, 49(1-3): 391-416 (2001); and        Well defined dye adsorbents for protein purification, J. Scoble and R. Scopes, J Mol Recognit, 9(5-6): 728-32 (1996).        
Use of dye-ligand affinity chromatography for the purification of polypeptides such as antibodies and proteins containing antibody fragments (for example, Fc fusion proteins) is a desirable method of choice, if such media can be used efficiently and effectively, because dye-ligand affinity chromatography is relative inexpensive, is resistant to chemical and biological degradation, and is suitable for in situ cleaning and sterilization.
The potential utility of dye-ligands for protein purification was discovered in the 1960's when scientists found that the enzyme pyruvate kinase co-eluted with Blue Dextran during a column chromatographic purification procedure. Blue Dextran was, in turn, subsequently used to purify pyruvate kinase from human erythrocytes. See, Hage, page 232.
Since the 1960's dyes have been used as ligands for purification of proteins such as albumin and other blood proteins, oxidoreductases, decarboxylases, glycolytic enzymes, nucleases, hydrolases, lyases, synthases, and transferases. See, Hage, page 232.
Dye-ligand chromatographic media is available from a number of commercial sources such as Sigma-Aldrich Inc., Amersham Pharmacia Biotech Corp., BioRad Laboratories Inc., and GE Healthcare Biosciences Corp. Examples of such media include: BLUE-SEPHAROSE®, CAPTO™ BLUE, AFFI-GEL™ Blue Agarose, CB3GA-Agarose, Blue-Trisacryl, Reactive Brown 10-Sepharose, Reactive Green 19-Sepharose, Reactive Red 120-Sepharose, and Reactive Yellow 3-Sepharose. Additional examples of dye molecules which may be attached to insoluble chromatographic support matrices include Cibacron Blue 3GA, Procion Red HE-3B, Procione Rubine MX-B, Procion Yellow H-A, and Turqouise MX-G See, Hage, page 233.
Triazine dyes are the most common molecules use in dye-ligand chromatography. The chemical structure of such molecules typically comprise a chromophore unit (the color producing moiety) joined through an amino-bridge to another organic molecule (such as 1,3,5-sym-trichlorotriazine) used for attachment to an insoluble support (such as agarose, dextran, or cellulose).
BLUE-SEPHAROSE™ or, more particularly, BLUE SEPHAROSE™ 6 Fast Flow is an affinity chromatography medium commercially available from GE Healthcare Bio-Sciences Corp. (Piscataway, N.J.). BLUE SEPHAROSE™ 6 Fast Flow is a chromatography medium comprising a bead structure of 6% highly cross-linked agarose to which Cibacron Blue 3G dye has been covalently linked at a density of approximately 7 micromols Cibacron Blue 3G 3G/mL of drained medium. GE Healthcare reports that “BLUE SEPHAROSE™ 6 Fast Flow is Cibacron Blue 3G covalently attached to the SEPHAROSE™ 6 Fast Flow “matrix by the triazine coupling method. The blue dye binds many proteins, such as albumin, interferon, lipoproteins and blood coagulation factors. It also binds several enzymes including kinases, dehydrogenases, and most enzymes requiring adenyl-containing cofactors e.g. NAD+. The highly cross-linked matrix provides a stable, rigid medium. Blue Sepharose Fast Flow belongs to the BIOPROCESS™ media family. BIOPROCESS™ media are separation media developed, made and supported for industrial scale—especially the manufacture of healthcare products. With their high physical and chemical stability, very high batch-to-batch reproducibility, and Regulatory Support File back-up, BioProcess media are ideal for all stages of an operation—from process development through scale-up and into production.” See, GE Healthcare Bio-Sciences Literature File No. 71-7055-00 AG (printed in Sweden by Wikströms, Uppsala, January 2006 (No. 1050991)); this literature file is hereby incorporated by reference solely to the extent that it does not contradict or conflict with the teachings in the present patent application; in the event that contradictions or conflicts exist, the present application is intended to supercede and/or take precedence over any such contradictory or conflicting material.
CAPTO™ BLUE is an affinity chromatography medium commercially available from GE Healthcare Bio-Sciences Corp. (Piscataway, N.J.). CAPTO™ BLUE is a chromatography medium comprising a bead structure of highly cross-linked agarose to which Cibacron Blue dye has been covalently linked at a density of approximately 11 to 16 micromols Cibacron/mL of medium. GE Healthcare markets CAPTO™ BLUE “for the capture of albumin, as well as purification of enzymes and recombinant proteins at laboratory and process scale.” GE Healthcare also reports that CAPTO™ BLUE was “[d]eveloped from Blue Sepharose™ 6 Fast Flow, [and] Capto Blue is more chemically stable and has a more rigid agarose base matrix than its predecessor. These improvements allow the use of faster flow rates and larger sample volumes, leading to higher throughput with no significant reduction in binding capacity.” See, GE Healthcare Bio-Sciences Data File No. 28-9392-46 AA (06/2008); this data file is hereby incorporated by reference solely to the extent that it does not contradict or conflict with the teachings in the present patent application; in the event that contradictions or conflicts exist, the present application is intended to supercede and/or take precedence over any such contradictory or conflicting material.
Optimal conditions for isolating a protein using a dye-ligand chromatography media are determined empirically depending on the specific target protein to be isolated. Variables likely to have significant effect on the ability to efficiently and effectively isolate or purify a protein include: pH; ionic strength of the solution; the composition of the mixture which the protein is in; temperature, sample size; and, of course, the specific dye-ligand and support matrix being used.
Purification of Antibodies Using Protein-A and Other Procedures
Protein-A affinity chromatography is one of the most widely used methods for the capture and isolation of monoclonal antibodies (mAbs) and Fc-fusion proteins. Protein-A is quite effective in removing host cell protein (HCP) and DNA while producing antibody mixtures of relatively high yield. Disadvantages to use of protein-A, however, include the binding and co-elution of PRIs with the target antibody, co-elution of aggregates, mis-folded antibody forms, and half-antibodies. Thereafter, following protein-A purification there is often considerable loss of antibody yield resulting from subsequent procedures (e.g., ion exchange and hydrophobic interaction chromatography methods) aimed at removing PRIs.
Other separation methods have also been routinely employed for the purification of antibodies. Such methods commonly rely on interactions based on charge (e.g., ion exchange), hydrophobicity (HIC; hydrophobic interaction chromatography), size (e.g., size exclusion chromatography), affinity (as with Protein-A) or combinations of such methods.
A patent application by Gagnon teaches use of PEG (or other aqueous soluble nonionic organic polymers) in mixed-mode chromatography antibody purification procedures. See, U.S. Patent Application Pub. No. 2008/0177048, filed Jan. 7, 2008, published Jul. 24, 2008 (hereinafter “Gagnon”). An important distinction of the present invention over Gagnon, however, is that Gagnon teaches that use of PEG enhances the binding capacity of antibodies, aggregates, and viruses on the various types of mixed-mode chromatography resins listed therein. In particular, Gagnon emphasizes the use of PEG throughout the chromatography purification steps as a means of enhancing the resin binding capacity (i.e., incorporation of PEG when antibody is first applied to the column and use of PEG in the wash and elution phases). Indeed, Gagnon teaches that “the presence of nonionic organic polymer preferentially enhances the retention of antibody on mixed mode chromatography supports in comparison to most contaminating proteins . . . ” See e.g., Gagnon at page 2, paragraph [0014] (emphasis added). Although Gagnon lists multiple types of available mixed mode chromatographic resins and types of interaction forces (see e.g., Gagnon paragraph [0003] and [0012]), the clear emphasis in Gagnon is on, and all of the examples were done with, hydroxyapatite chromatography resins. More particularly, Gagnon does not include any mention of dye-ligand chromatography resins, in general, or blue-dye chromatography resins, in particular. Moreover, in contrast to the “enhanced retention” of antibodies on mixed-mode resins in the presence of organic polymers taught in Gagnon, the present invention pertains to use of organic polymers (such as PEG) to specifically elute target antibodies from dye-ligand chromatography resins by incorporating organic polymers (such as PEG) only in the elution phase (such as in a single-step or gradient-elution phase). In other words, with dye-ligand chromatography resins, organic polymers such as PEG are not used to enhance binding of antibodies to the resin, but are instead used to elute target antibodies from the resin. Indeed, this distinction may explain the comment in Gagnon that “Mixed mode chromatography supports provide unique selectivities that cannot be reproduced by single mode chromatography methods such as ion exchange, however method development is complicated, unpredictable, and may require extensive resources. Even then, development of useful procedures may require long periods of time, as exemplified by hydroxyapatite.” See, Gagnon, paragraph [0004].